System and method of measuring molecular interactions

ABSTRACT

The present disclosure relates to a device for measuring surface plasmon resonance and fluorescence of a sample, a system for determining the rate of catalytic activity of an enzyme, a method of determining the rate of catalytic activity of an enzyme, and a method of measuring the adsorption and reactivity of a substance, all of which use SPR and SPEF methods simultaneously. This invention also relates generally to systems and methods for measuring diffusion and reactivity of macromolecules on a surface.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to systems and methods for measuringmolecular interactions. More specifically, the invention relates tomeasurements of surface adsorption and/or reactions using a combinationof surface plasmon resonance and surface plasmon enhanced fluorescencedetection. This invention also relates to systems and methods formeasuring diffusion and reactivity of macromolecules on a surface.

2. Description of the Related Art

Surface Plasmon Resonance (SPR) is a physical process that occurs whenlight of a particular wavelength that has been plane polarized(p-polarized) interacts with a thin metal film at a specific angle ofincidence. One way to couple light to a thin metal film is through aprism of glass or other optically transparent solid. Light directedthrough glass or another optically transparent solid such as quartz orplastic and impinging on an interface of the solid and dielectric, willpartially reflect back out of the solid and otherwise refract into thedielectric at the point of impingement. When the angle of incidence ofthe incoming light achieves a critical angle all of the light willreflect back through the solid. This is known as total internalreflection (IIR), and even though no photons are passing beyond thesurface of the solid, an electrical field from the photons, known as anevanescent wave, extends beyond the reflecting surface.

The thin film of metal used in SPR contains free electron constellationsthat are affected by electrical fields. When such a film is applied tothe interface between a clear solid and a dielectric, the energy fieldof the photons causes excitation of these electron constellationsresulting in the propagation of surface plasmons, or electron densitywaves. These waves propagate along the surface of the thin film. Thepropagation of the photon electrical field is dependent on the frequencyand the angle of the incident light, as well as the refractive indicesof the dielectrics on either side of the film.

When p-polarized light of the correct frequency strikes the surface atthe appropriate angle under TIR, the energy of the photon energy fieldis transferred to the surface plasmons as resonance is achieved. Surfaceplasmon resonance occurs when the component of the light wave-vectormatches the real component of the wave-vector of the surface plasmon.This resonance results in a sharp decrease in the energy of thereflected light as that energy is transferred to the plasmons.

As mentioned previously, the angle and energy at which resonance occursis dependent on the refractive properties of the dielectrics on eitherside of the thin film. For a monochromatic light source, the angle ofincidence at which resonance occurs will vary if the properties of thesurface of the film change due to its altered refractive index.Similarly, if a sample to be measured is layered on the thin film, thedielectric properties at the film surface change and the angle of SPRchanges accordingly. Because of this phenomenon, the angle of incidenceis a direct measure of the characteristics at the surface of the thinfilm.

From these properties, various reactions of the thin film can bedetected and measured by the varied reflective energy of the reflectedlight. For instance, the adsorption of various organic compounds can bemeasured on the surface of the thin film.

For example, the diffusion of proteins once the proteins are adsorbed tothe surface can be measured. Bovine serum albumin (BSA) adsorbed to asection of a glass cover slip, while exposing the remainder of the coverslip only to protein-free buffer, advanced with the passage of time(Dt)^(1/2) with a dependence consistent with a diffusion typephenomenon. Diffusion across a gradient (a region in which theconcentration of a substance changes over distance) could be describedby a Fickian analysis (Transport Phenomena, R. Byron Bird, Warren E.Stewart, & Edwin N. Lightfoot, John Wiley & Sons, New York, 2^(nd)Edition, 2002). The self-diffusion coefficient of BSA on a quartzsurface using fluorescence recovery after patterned photobleaching(FRAPP) techniques has been determined (Burghardt, T. P.; Axelrod, D.,Biophys. J., 1981, 33, 455467). The results have been confirmed andextended to polymer surfaces (Tilton, R. D.; Robertson, C. R.; Gast, A.P. J. Colloid Interface Sci. 1990, 137, 192-203). Surface diffusion inother systems such as membrane proteins (Scallettar, B. A.; Abney, J.R.; Owicki, J. C., Proc. Natl. Acad. Sci. U.S.A. 1988, 85(18),6726-6730; Abney, J. R.; Scallettar, B. A.; Owicki, J. C., Biophys. J.,1989, 55(5), 817-833; Abney, J. R.; Scallettar, B. A.; Owicki, J. C.,Biophys. J., 1989, 56(2), 315-326), proteins adsorbed to the surface ofchromatography resins (Ma, Z; Whitley, R. D.; Wang, N. H. L., AIChE J.,1996, 42(5), 1244-1262; Chen, W. D.; Dong, X. Y.; Sun, Y., J.Chromatogr. A, 2002, 962(1-2), 29-40), and DNA oligonucleotides atsolid/liquid interfaces (Chan, V.; Graves, D. J.; Fortina, P.; Mckenzie,S. E., Langmuir, 1997, 13(2), 320-329; Chan, V.; Mckenzie, S. E.;Surrey, S; Fortina, P.; Graves, D. J., J. Colloid Interface Sci. 1998,203(1), 197-207) has been studied.

Many varied techniques including forced Rayleigh techniques (Antonietti,M.; Coutandin, J.; Grütter, R.; Sillescu, H. Macromolecules 1984, 17,798; Ehlich, D.; Takaneka, M.; Hashimoto, T., Macromolecules, 1993,26(3), 492498) and NMR (Foy, B. D.; Blake, J., Journal of MagneticResonance, 2001, 148(1), 126-134) have been used to probe proteinlateral mobility, but the most prevalent experimental tool has beenFRAPP or the related procedure, TIR-FRAPP (total internalreflection—fluorescence recovery after patterned photobleaching). FRAPPentails working with a fluorescently labeled protein (or othermacromolecule) adsorbed to a surface. A high intensity pulse of laserlight is shined on sections of the surface photobleaching allchromophores within those regions. Any recovery of fluorescenceintensity in the photobleached areas can be correlated to surfacediffusion of the adsorbed fluorescently labeled proteins. FRAPP resultsare highly reproducible, and it appears FRAPP effectively measuressurface diffusion on a wide variety of materials. TIR-FRAPP allows oneto illuminate molecularly thin surface layers of fluroescent materialusing total internal reflection of an incident laser beam. As such, theillumination source does not interact with fluroescent materials in thebulk and restricts interrogation to the very near surface regions. Thisfeature enhances the surface sensitivity of the measurement.

The main problems with FRAPP related techniques are that a protein mustbe labeled, which can lead to artificial adulteration of its threedimensional structure, that FRAPP cannot effectively probe gradientdiffusion (i.e. the movement of a substance from a region of high to aregion of low concentration) and that FRAPP requires the protein to beirreversibly adsorbed to the surface. FRAPP cannot distinguish betweenpure surface diffusion and protein desorption followed by subsequentbulk diffusion and reattachment at a different spot on the surface ifthe protein is weakly adsorbed such that there is some exchange with thebulk.

Surface diffusion plays an important role in the interaction of enzymeswith biopolymers in the β-amylase starch gel system. Surface diffusion,rather than adsorption or intrinsic reactivity, is the deciding factorin proteolytic cleavage of substrate surfaces (Brode, P. F.; Erwin, C.R.; Rauch, D. S.; Barnett, B. L.; Armpriester, J. M.; Wang, E. S. F.;Rubingh, D. N., Biochemistry 1996, 35, 3162-3169). TIR-FRAPP can be usedto measure the surface diffusion of collagenase irreversibly adsorbed toand reacting with a collagen surface (Gaspers, P. B.; Robertson, C. R.;Gast, A. P., Langmuir 1994, 10, 2699-2704). FRAPP can be used toquantify the surface diffusion rate of cellulase interacting with acellulose surface (Jervis, E. J.; Haynes, C. A.; Kilburn, D. G., J.Biol. Chem. 1997, 272(38), 24016-24023). In each of the above cases, theenzyme was adsorbed to the surface so that there was little or noexchange with the bulk, thus making the FRAPP results reliable.

Microfluidic systems can be made to miniaturize traditional benchtopoperations into miniature lab-on-a-chip type systems. Assays as variedas enzyme kinetics (Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.;Jacobson, S. C.; Ramsey, J. M., Anal. Chem. 1997, 69, 3407-3412; Duffy,D. C.; Gillis, H. L.; Lin, J.; Sheppard, N. F.; Kellogg, G. J., Anal.Chem. 1999, 71, 4669-4778; Hadd, A. G.; Jacobson, S. C.; Ramsey, J. M.,Anal. Chem. 1999, 71, 5206-5212), capillary electrophoresis (Khandurina,J.; McKnight, T. E.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.;Ramsey, J. M., Anal. Chem. 2000, 72, 2995-3000), immunoanalysis (Hatch,A.; Kamholz, A. E.; Hawkins, K. R.; Munson, M. S.; Schilling, E. A.;Weigl, B. H.; Yager P. A., Nature Biotechnology 2001, 19, 461-465;Eteshola, E.; Leckband, D., Sensors and Actuators B-Chemical 2001, 72,129-133; Cheng, S. B., Anal. Chem. 2001, 73, 1472-1479; Yang, T. L.;Jung, S. Y.; Mao, H. B.; Cremer, P. S., Anal. Chem. 2001, 73, 165-169),isoelectric focusing (Macounova, K.; Cabrera, C. R.; Holl, M. R.; Yager,P., Anal. Chem. 2001, 72, 3745-310; Macounova, K.; Cabrera, C. R.;Yager, P., Anal. Chem. 2001, 73, 1627-1633) and others have beenperformed on microfluidics platforms. The basis for microfluidictechniques is in fact a very simple outcome of low Reynolds numberhydrodynamics. The Reynolds number is a dimensionless quantity definedas: ${Re} = \frac{\rho\quad{UD}}{\mu}$

where ρ is the fluid density, U the average velocity, D a characteristiclength for the system and μ the fluid viscosity (Transport Phenomena, R.Byron Bird, Warren E. Stewart, & Edwin N. Lightfoot, John Wiley & Sons,New York, 2^(nd) Edition, 2002). The Reynolds number also represents aratio of inertial to viscous forces. In a microfluidic system, since allsystem dimensions are small, the Reynolds number is low. Accordingly,the fluid essentially has no inertia and acts as though it were“massless.” Such properties of fluid flow may be used according to theprinciples of the current invention.

A primary consequence of the low Reynolds flows is that multiple fluidstreams can be made to flow side-by-side one another with minimal mixingbetween or among these streams. Indeed, the only mixing of componentscan be due to diffusion across the boundaries separating the multiplefluid streams. This diffusional mixing in turn can be minimized byflowing the streams at high velocities while at the same time notviolating the constraint of maintaining a low Reynolds number.

As an example of this, a low Reynolds number phenomena has been used tomeasure a variety of molecular phenomena within the interdiffusionregions (i.e. the regions where diffusional mixing is occurring) atfluid-fluid interfaces in an embodiment known as the T sensor (Kamholz,A. E.; Weigl, B. H.; Finlayson, B. A.; Yager, P., Anal. Chem. 1999,71(23), 5340-5347; Ismagilov, R. F.; Stroock, A. D.; Kenis, P. J. A.;Whitesides, G. M.; Stone, H. A.,

Appl. Phys. Lett. 2000, 76(17), 2376-2378). The width of thisinterdiffusion region can be simply calculated by the relation:x _(rms)=√{square root over (2Dt)}

where x_(rms) is the root mean squared distance traveled by a moleculein a time t with diffusion coefficient D (Kamholz, A. E.; Weigl, B. H.;Finlayson, B. A.; Yager, P., Anal. Chem. 1999, 71(23), 5340-5347). Thetime t is thus given by a characteristic time that is dependent on thedistance along the channel (L) and the flow velocity (U) by the relation$t = \frac{L}{U}$

rather than an actual time. The use of low Reynolds number flow topattern surfaces has been described (Takayama, S.; McDonald, J. C.;Ostuni, E.; Liang, M. N.; Kenis, P. J. A.; Ismagilov, R. F.; Whitesides,G. M., Proc. Natl. Acad. Sci. U.S.A. 1999, 96(10), 5545-5548; Kenis, P.J. A.; Ismagilov, R. F.; Whitesides, G M, Science 1999, 285, 83-85;Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M.,Anal. Chem. 1998, 70(23), 4974-4984). The relations change somewhat nearthe surface (diffusion distance follows a (DHt)^(1/3) rather than a(Dt)^(1/2) dependence) but the same principles hold (Takayama, S.;McDonald, J. C.; Ostuni, E.; Liang, M. N.; Kenis, P. J. A.; Ismagilov,R. F.; Whitesides, G. M., Proc. Natl. Acad. Sci. U.S.A. 1999, 96(10),5545-5548). For example, a three inlet channel to pattern E. coli cellsin alternating stripes on a substrate surface has been shown (Takayama,S.; McDonald, J. C.; Ostuni, E.; Liang, M. N.; Kenis, P. J. A.;Ismagilov, R. F.; Whitesides, G. M., Proc. Natl. Acad. Sci. U.S.A. 1999,96(10), 5545-5548).

Soft lithography is a popular, reliable technique for producingmicrofluidic devices (Duffy, D. C.; McDonald, J. C.; Schueller, O. J.A.; Whitesides, G. M., Anal. Chem. 1998, 70(23), 4974-4984). It makesuse of an elastomeric material known as polydimethylsiloxane (PDMS) thatis both flexible enough to seal against most types of substrate surfacesand rigid enough to maintain channel structures. The techniqueincorporates photolithography methods that have been used for years inthe semiconductor industry into a micromolding scheme that eventuallyproduces micron sized structures in negative relief in PDMS (see, Duffy,D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M., Anal.Chem. 1998, 70(23), 4974-4984 and for example, FIG. 2).

Initially, a clean dry silicon wafer is spin coated with a layer ofphotoresist (typically SU8, Microchem Inc.) equal to the desiredthickness of the microchannel. A mask containing the pattern ofmicrofluidic structures is then placed over the wafer and exposed toultraviolet light. Because photoresist is photosensitive, it willcrosslink in the areas exposed to the light and thus when submerged indeveloper solution, only the exposed regions of photoresist remain onthe wafer. This pattern of photoresist thus serves as the mold for thenext micromolding step. A PDMS prepolymer along with a curing agent arethen cast on the pattern containing wafer. At an elevated temperature,the PDMS cures, producing a soft flexible material with the channelsembedded in negative relief. The PDMS is peeled back from the wafer andthen sealed against the substrate of choice. Holes are made at theinlets and outlets of the PDMS piece to allow delivery of reagents toand from the newly formed microfluidic chip.

Thus, what is needed in the art is a system that can measure multiplemolecular interactions quickly and efficiently. What is also needed inthe art is a method for making use of microfludics to pattern areas ofthe substrate surface that are accessible to an enzyme.

SUMMARY OF THE INVENTION

Disclosed is a device for measuring surface plasmon resonance andfluorescence of a sample, comprising a light source capable of directinga beam of light at a sample cell, where the sample cell comprises afirst compound bound to a metallic surface; a first detector formeasuring the surface plasmon resonance from the metal surface; a seconddetector for measuring the fluorescence intensity from the surface; anda module for calculating the combined adsorption of two species from thesurface plasmon resonance measurement and the fluorescence intensitymeasurement.

Also disclosed is a system for quantifying the properties of molecules.Specifically, an aspect disclosed is a system for determining the rateof surface catalytic activity of an enzyme; comprising a light sourcecapable of directing a beam of light at a sample cell, where the samplecell comprises a fluorescent compound bound to a metallic surface; afirst detector for taking a surface plasmon resonance measurement of thesurface in the sample cell as an enzyme is contacted with the compound;a second detector for taking a fluorescence intensity measurement of thecompound as the enzyme is contacted with the compound; and a module forcalculating the catalytic activity of the enzyme from the surfaceplasmon resonance measurement and the fluorescence intensitymeasurement.

In addition, a method is disclosed for determining the rate of catalyticactivity of an enzyme, comprising providing a sample cell comprising afluorescently labeled compound is bound to a metallic surface; flowingan enzyme sample through the sample cell; measuring the surface plasmonresonance of the sample cell over time to determine the amount of theenzyme that is bound to the first fluorescently labeled compound;measuring the fluorescence of the first fluorescently labeled compoundover time to determine the amount of the first fluorescently labeledcompound bound to the metallic surface; and calculating the catalyticactivity of the enzyme from the surface plasmon resonance measurementand the fluorescence intensity measurement.

Furthermore, disclosed is a method of measuring the adsorption ofmultiple substances, comprising a) exposing said first substance on athin metal layer of a top window of a sample cell to a first beam oflight from a light source; b) detecting a reflection of the first beamof light with a reflectivity detector, thereby taking a surface plasmonresonance measurement; c) detecting a fluorescence light from the firstsubstance with a fluorescence intensity detector; d) contacting a secondsubstance with the first substance to provide a mixture; e) exposing themixture to a second beam of light from a light source; f) detecting areflection of the second beam of light with a reflectivity detector,thereby taking a surface plasmon resonance measurement; g) detecting afluorescence light from the mixture with a fluorescence intensitydetector; and h) comparing the results of the detections of steps b) andc) with the detections of steps e) and g).

Additionally disclosed is a device for measuring diffusion andreactivity comprising a surface for flowing at least two interfacingfluid streams and for creating and relaxing surface gradients in the atleast two fluid streams, at least one stream containing macromolecules,the macromolecules interacting with the surface, wherein the flow has alow Reynolds number so that the at least two fluid streams do not mix;and a detector. The at least two interacting streams can be three orfive streams. More streams can be envisioned. The device is particularlysuited for use with macromolecules. Furthermore, the device can bepracticed wherein the detector comprises fluorescence microscopy,plasmon imaging, ellipsometric imaging, brewster angle microscopy, totalinternal reflection microscopy, FRAPP or a combination of any of theabove. The surface of the device may comprise surfaces that areplastics, polymers, SAMS (self-assembled monolayers), lipid bilayers,glass, transparent materials, reflective materials, gold, biomaterialsor biodegradable materials.

Finally, disclosed is method for measuring diffusion and reactivitycomprising flowing at least two interfacing fluid streams on a surface,at least one stream containing macromolecules, the macromoleculesinteracting with the surface, wherein the flow has a low Reynolds numberso that the at least two fluid streams do not mix; creating and relaxingsurface gradients as a result of flowing of the at least two fluidstreams and detecting diffusion and reactivity. The at least twointeracting streams can be three or five streams. More streams can beenvisioned according to the invention. The method is particularly suitedfor use with macromolecules. Furthermore, the method can be practicedwherein the detecting step further comprises fluorescence microscopy,plasmon imaging, ellipsometric imaging, brewster angle microscopy, totalinternal reflection microscopy, FRAPP or a combination of any of theabove. The surface may comprise surfaces that are plastics, polymers,SAMS, lipid bilayers, glass, transparent materials, reflectivematerials, gold, biomaterials or biodegradable materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an embodiment of a system that can be usedfor conducting multiple tests simultaneously and recording and analyzingany corresponding test results.

FIG. 2 is a schematic view of a test apparatus of the system of FIG. 1.

FIG. 3 is a schematic view of a sample cell of the apparatus of FIG. 2.

FIG. 4 is a schematic view of the modules of a computer that can beimplemented in the system of FIG. 1.

FIG. 5 is a flow chart of a process that can be used to normalizemeasured values of reflectance in the system of FIG. 1.

FIG. 6 shows (a) a typical surface plasmon spectrum for the interfacebetween gold (n=0.17216, k=3.4218) and 2 mM carbonate buffer (n=1.335).(b) Shift to a higher resonance angle in the surface plasmon curve uponformation of BSA monolayer (n=1.57). The arrow indicates the angle ofmaximum vertical resolution (56.5°).

FIG. 7 shows a sample surface plasmon signal before (a) and after (b)normalization by the reference gold-air signal. A substantialimprovement in the surface plasmon image quality is observed.

FIG. 8 depicts a biotin/avidin binding experiment. In (a), a monolayerof labeled avidin is bound to a biotin surface. In (b), an incompletemonolayer of labeled avidin is formed followed by completion of thelayer by unlabeled avidin. In (c), the first avidin layer is leftunlabeled and another layer of fluorescently labeled biotin-BSA is boundto the avidin surface.

FIG. 9 shows the results of the first experiment described in FIG. 8 a.The graph in (a) depicts the time course of the experiment. The insetshows the linear correlation between the two signals.

FIG. 10 shows (a) an incomplete monolayer experiment in which firstfluorescent avidin is flowed over the biotin surface and thennon-fluorescent avidin. No rise in SPEF is seen upon addition of theunlabeled avidin. (b) The corresponding control experiment in whichfluorescently labeled avidin has been added at both steps.

FIG. 11 shows the results of (a) a biotin/avidin/BSA sandwich experimentin which only BSA is fluorescently labeled. The SPEF signal only risesin the second step.

The inset shows the amounts of BSA and avidin present on the surfaceseparately (b) Corresponding control experiment in which neithercomponent is labeled.

FIG. 12 shows the results of a PMSF inhibited enzyme experiment. Arrowsindicate time of addition. The displacement in the signal followingaddition of inhibited enzyme indicates enzyme adsorption. The decreasein SPR signal after addition of active enzyme confirms the presence ofBSA on the surface.

FIG. 13 shows channel geometry where two fluids are flown together.

FIG. 14 shows steps used in soft lithography.

FIG. 15 shows channel geometry where fluids are flown with fluorescentBSA as the surface substrate. The figure shows that initially athickness profile of the surface will be flat when two fluids are flowntogether. Enzyme solution in the bulk is localized to the middle lane,and any widening of the trench is due to surface diffusion as it reactsaway surface bound protein.

FIG. 16 shows a flow scheme according to an embodiment of the invention.

FIG. 17 shows a five lane apparatus with a negative pressure “pull”configuration.

FIG. 18 shows the results of a three lane experiment with enzyme floweddown the middle lane. FIG. 18 a shows the results, with the beginning ofthe trench, caused by enzymatic erosion of the surface, visible. FIG. 18b shows intensity profiles across the channel. After two hours of flow,considerable widening of the trench can be seen.

FIG. 19 shows the results of a five lane experiment with the resultsflowed down the middle. FIG. 19 a shows flaring out; FIG. 19 b shows thealteration of the structure of the junction to overcome the flaring out,as shown in FIG. 19 b; FIG. 19 c shows the is improvement as a result ofthe improved channel.

FIG. 20 shows the results of a five lane assay using subtilisin and aG100R mutant. The positive mutant is seen to be slower reacting and lessmobile on the surface than the corresponding wildtype subtilsin enzyme.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Embodiments of the invention will now be described with reference to theaccompanying figures, wherein like numerals refer to like elementsthroughout. The terminology used in the description presented herein isnot intended to be interpreted in any limited or restrictive mannersimply because it is being utilized in conjunction with a detaileddescription of certain specific embodiments of the invention.Furthermore, embodiments of the invention may include several novelfeatures, no single one of which is solely responsible for its desirableattributes or which is essential to practicing the inventions hereindescribed.

One embodiment of the invention is a system and method forsimultaneously measuring surface plasmon resonance and surface plasmonenhanced fluorescence during a molecular interaction. As is discussed indetail below, a molecular interaction includes interactions between anyspecies of chemical, biological or other compound. As used herein,“molecular” includes inorganic and organic chemical compounds, proteins,peptides, antibodies, antigens, enzymes and the like. Advantageouslysimultaneous measurement of both SPR and SPEF permits researchers tomeasure two or more species or properties of the molecular interactionat the same time. For example, an enzymatic substrate can befluorescently labeled and bound to a thin film surface. An SPRmeasurement can then be taken to reveal the quantity of substrate boundto the surface. After the quantity is determined, an appropriate enzymecan be added to the reaction and a measurement of the SPR and SPEF ofthe reaction over time can be taken. Because the SPEF will measure theamount of fluorescently labeled substrate in the reaction, and the SPRwill determine the amount of enzyme bound to substrate at the surface ofthe thin film, a reaction rate can be calculated. Of course, this isonly one example of the type of complex molecular interaction that canbe measured by embodiments of the invention. The system and methodsdescribed herein allow new testing methods to determine the reactionkinetics of one or more compounds where the compounds have differentproperties that can each independently vary the SPR outcome, the SPEFoutcome or both. For instance, two compounds, one of them beingfluorescent, can be reacted in a mixture. SPR can then be used tomeasure the reactions of both, while SPEF only measures the reaction ofthe fluorescent material. By taking the difference between the SPRmeasurement and the SPEF measurement, the reaction rate of thenon-fluorescent material can be determined.

Another embodiment is a system and method for measuring diffusion andreactivity of macromolecules on a surface. This aspect, for example,allows direct observation of the lateral diffusivity of the enzyme as itcleaves substrate from the surface and allows observation of theproperties without adulteration of the enzyme structure by, for example,labeling (e.g., fluorescent and radioactive labeling). Specificembodiments include a microfluidic patterning technique for thelabel-free measurement of an enzyme's lateral diffusivity as itinteracts with a substrate surface.

Another embodiment is drawn to a method of enhancing the performance ofenzymes such as cellulase, amylase, protease, polyesterase, lipase,mannanase, cutinase, oxidases etc that reacts with substrates that arebound to a surface or solid substrates. Proteolytic removal of a proteinfrom a given surface will depend on the enzyme adsorption to thesubstrate surface, diffusion of the adsorbed enzyme on the substratesurface and the inherent proteolytic activity of the enzyme. A proteasethat adsorbs to the surface but cannot diffuse on the surface will notbe very efficient in removing the protein from the surface. Similarly aprotease that does not adsorb to the surface will not be very efficientin removing the protein from the surface. Therefore, there is an optimumadsorption for optimal performance (removal of protein from the surface)that is between 20% and 80% of the protease in solution (especiallyuseful for applications that use low enzyme concentrations). In the sameway, there is an optimal diffusion that is required for performancewhich is greater than 10% but less than 60% of the theoretical maximumfor a molecule that does not have any specific interaction with theprotein surface. Therefore protease molecules of a given proteolyticactivity with the above mentioned limits of adsorption and diffusionwill yield the maximum protein removal from the substrate surface. Oncethe adsorption and diffusion are not limiting the performance, then, theinherent proteolytic activity can be improved. This cycle can berepeated until the desired performance is achieved.

I. Aspects of the Invention

Disclosed is a system for quantifying the properties of molecules.Specifically, in a first aspect, embodiments of the present inventionrelate to a device for measuring surface plasmon resonance andfluorescence of a sample, comprising a light source capable of directinga beam of light at a transparent prism bound to a metallic surface; afirst detector for measuring the surface plasmon resonance of themetallic surface; a second detector for measuring the fluorescenceintensity from the sample; and a module for calculating the adsorptionor catalytic activity of the enzyme from the surface plasmon resonancemeasurement and the fluorescence intensity measurement.

In certain embodiments, the metallic surface of the above device isformed over a piece of glass. The metallic surface may be made up of onesingle metal, or may comprise two or more different metals in the formof alloys. In some embodiments, a second layer of metal may be placedbetween the glass surface and the metallic surface, where the two layersof metal are made up of different types of metals.

In certain embodiments, the metallic surface may comprise a transitionmetal. A “transition metal” is a metal within columns 3-12 of theperiodic table. Some main group metals are also suitable for use as themetallic surface. Some of the metals contemplated within the scope ofthe invention include scandium, titanium, vanadium, chromium, manganese,iron, cobalt, nickel, copper, zinc, gallium, yttrium, zirconium,niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver,cadmium, indium, tin, lanthanum, hafnium, tantalum, tungsten, rhenium,osmium, iridium, platinum, gold, mercury, thalium and lead. The metalmay be in its elemental form or in a compound form. In some embodiments,the metal is selected from the group consisting of gold, silver,mercury, titanium, which may be in titanium dioxide form and copper. Inother embodiments, the metal is gold.

In certain embodiments, the first compound is attached directly to themetallic surface. In other embodiments, the first compound may beattached to a linker arm, which in turn is attached to the metallicsurface. In some embodiments, the linker arm may form a monolayer, suchas a self-assembled monolayer. The monolayer may then comprise afunctional group to which the first compound can be attached. Methods offormation of self-assembled monolayers on metallic surfaces is known tothose of skill in the art. See, for example, Nuzzo, R. G.; Allara, D. L.J. Am. Chem. Soc. 1983, 105, 448; Bain C. D.; Troughton, E. B.; Tao,Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc.1989,111,321-335; and Ulman, A. Chem. Rev. 1996, 96, 1533; all of whichare incorporated by reference herein in their entirety, including anydrawings.

In some embodiments, the sample compound is an enzyme and the firstcompound is a substrate for the enzyme, while in other embodiments thefirst compound is an enzyme and the sample compound is a substrate forthe enzyme.

Embodiments of the invention include those in which the sample compoundis contacted with the first compound. In some embodiments, neat samplecompound, i.e., sample compound that is not dissolved in any solutionsor diluted in any way, is added directly to the metallic surface, towhich the first compound is attached.

In other embodiments, the sample compound is mixed with a carrier. Thecarrier, and the sample compound contained therein, are then passed overthe metallic surface comprising the first compound. The carrier may be aliquid carrier or a gas. If the carrier is a liquid, then the carrierand the sample compound form a solution and the solution is passed overthe metallic surface. If the carrier is a gas, then the carrier and thesample compound form a gaseous mixture, which is then passed over themetallic surface. In some embodiments, the carrier is a liquid. Incertain embodiments, the carrier is an aqueous solution. In otherembodiments, the carrier comprises an organic solvent. Thus, in certainembodiments, the solution comprising the sample compound flows throughthe sample cell, thereby causing the first compound and the samplecompound to come into contact with each other. This would work for otherapplications beyond enzymes.

In some embodiments the first detector is a reflectance detector, whichdetects light reflected from the sample cell. In certain embodiments thefirst detector is a charge couple device, or CCD, detector.

In some embodiments, the first detector measures the surface plasmonresonance of the metallic layer as a sample compound is contacted withthe first compound. In certain embodiments, the second detector measuresthe fluorescence intensity from the sample cell as the sample compoundis contacted with the first compound.

In certain embodiments, the angle the reflected light makes with thenormal of the sample cell is substantially equal to the angle the beamof light from the light source makes with the normal of the prism. Insome embodiments, the sum of the angle the reflected light makes withthe normal of the prism and the angle the beam of light from the lightsource makes with the normal of the prism is less than 180°. In theseembodiments the first detector may detect any variations in the anglethe reflected light makes with the normal of the prism.

Embodiments of the invention include those in which the second detectoris a fluorescence detector which detects fluorescence from the samplecell. The second detector may be located on the opposite side of thesample cell as the light source.

In some embodiments, the first compound fluoresces after beingilluminated by the light, whereas in other embodiments the samplecompound fluoresces after being illuminated by the light.

The light used in some embodiments of the invention may bemonochromatic. The light source used may be a laser light source.

In certain embodiments, the device further comprises at least onemirror, at least one prism, at least one collimator, or at least onelens, or a combination thereof, between the light source and the samplecell.

In certain embodiments, the module comprises a microprocessor. Themodule may also comprise a memory. Additionally, the module may comprisea set of computer implemented instructions.

In another aspect, the present invention relates to a system fordetermining the rate of catalytic activity of an enzyme; comprising alight source capable of directing a beam of light at a prism, where theprism is bound to a fluorescent compound bound to a metallic surface; afirst detector for taking a surface plasmon resonance measurement of thesample cell as an enzyme is contacted with the compound; a seconddetector for taking a fluorescence intensity measurement of the compoundas the enzyme is contacted with the compound; and a module forcalculating the catalytic activity of the enzyme from the surfaceplasmon resonance measurement and the fluorescence intensitymeasurement.

In a further aspect, the invention relates to a method of determiningthe rate of catalytic activity of an enzyme, comprising providing asample cell comprising a fluorescently labeled compound bound to ametallic surface; flowing an enzyme sample through the sample cell;measuring the surface plasmon resonance of the sample cell over time todetermine the amount of the enzyme that is bound to the firstfluorescently labeled compound; measuring the fluorescence of the firstfluorescently labeled compound over time to determine the amount of thefirst fluorescently labeled compound bound to the metallic surface; andcalculating the catalytic activity of the enzyme from the surfaceplasmon resonance measurement and the fluorescence intensitymeasurement.

In another aspect, the invention relates to a method of measuring theadsorption of multiple substances, comprising a) exposing the firstsubstance on a thin metal layer of a top window of a sample cell to afirst beam of light from a light source; b) detecting an intensity of areflection of the first beam of light with a light detector, therebytaking a surface plasmon resonance measurement; c) detecting afluorescence light from the first substance with a fluorescenceintensity detector; d) contacting a second substance with the firstsubstance to provide a mixture; e) exposing the mixture to a second beamof light from a light source; f) detecting a reflection of the secondbeam of light with a reflectivity detector, thereby taking a surfaceplasmon resonance measurement; g) detecting a fluorescence light fromthe mixture with a fluorescence intensity detector; and h) comparing theresults of the detections of steps b) and c) with the detections ofsteps e) and g).

In another embodiment, the invention is directed to a device and methodfor measuring diffusion and reactivity of macromolecules on a surface.This embodiment, for example, allows direct observation of the lateraldiffusivity of the enzyme as it cleaves substrate from the surface,which permits observation of properties without adulteration of theenzyme structure by, for example, labeling (e.g., fluorescent,radioactive, etc.). Specific embodiments include a microfluidicpatterning technique for the label-free measurement of an enzyme'slateral diffusivity as it interacts with a substrate surface.

In another embodiment, the invention is directed to a method foridentifying enzymes that perform optimally on substrate surfaces and theoptimized enzymes.

Since the above methods are practiced using the devices describedherein, all of the various embodiments described herein with respect tothe disclosed devices similarly apply to the disclosed methods andprocedures.

II. Overview

Certain systems and methods of the present invention are particularlysuited for measuring multiple variables in a molecular reaction at thesame time. The mechanism of many such reactions comprise multiple steps.Usually, one of the steps is slower than others and is known as therate-determining step. Step(s) before the rate-determining step arenormally in equilibrium, called “pre-equilibrium.” By knowing the rateconstants of the forward and reverse reactions in the pre-equilibrium,and the rate constant of the rate-determining step, the overall rate lawfor the reaction can be determined.

In certain embodiments of the present invention, a first compound isaffixed to a thin metal layer on a glass slide. The first compound canbe an enzyme, a protein, a substrate for an enzymatic reaction, or oneof the reactants of a reaction. In some embodiments disclosed herein,the first compound is called “the sample compound” or “the firstcompound.”

The first compound may be fluorescent. Its fluorescence may be inherent,i.e., the first compound itself fluoresces, or a fluorescent substituentmay be attached to the first compound prior to its affixation to theglass slide. The first compound may have more than one fluorophoreattached to it, thus providing the ability to measure its fluorescenceat multiple wavelengths. In other embodiments, the first compound is notfluorescent.

There are various ways the first compound may be affixed to the glassslide. The first compound may be made to react directly with the thinmetal layer. Thus, for example, a self-assembled monolayer of the firstcompound can be formed on the thin metal layer. In other embodiments,the first compound may be able to react with a functional group on ananchor attached to the thin metal layer. Thus, for example, aself-assembled monolayer comprising a functional group can be attachedto the thin metal layer, forming an anchor, and then the first compoundis made to react with that functional group, thereby being affixed tothe glass slide through the anchor. Alternatively, the first and secondcompound can be adsorbed simultaneously as well.

SPR and SPEF measurements are made of the glass slide while the firstcompound is affixed thereto. These measurements determine the baselinefor the measurements. The SPR measurement determines the thickness ofthe chemical layer on the thin metal layer and the SPEF measurementdetermines the maximum fluorescence of the sample prior to any reactiontaking place. Alternatively, the SPEF can measure the minimumfluorescence of the sample prior to any reaction taking place. A changein either or both of these measurements will then determine the extentof reaction involving either the thickness of the layer or the amount offluorescence present.

A second compound, also referred to herein as “the test compound,” isthen made to react with the first compound. The second compound may bean enzyme whose substrate is the first compound, may be a substrate forthe first compound enzyme, or may be another reactant in the reactionbeing studied. The second compound may or may not be fluorescent. Iffluorescent, its fluorescence may be inherent, i.e., the second compounditself fluoresces, or a fluorescent substituent may be attached to thesecond compound prior to the reaction taking place. The second compoundmay have more than one fluorophore attached to it, thus providing theability to measure its fluorescence at multiple wavelengths.

When the second compound binds to the first compound in the course ofthe reaction, the thickness of the chemical layer on the thin metallayer changes. The layer becomes thicker. This increase in the thicknessaffects the measurement obtained by the SPR.

When the first and second compounds react, the fluorescence of thesample changes. The reaction between the two may be a cleaving reaction,meaning the first compound cleaves a part of the second compound, orvice versa. If the reaction is a cleaving reaction, then thefluorescence of the sample decreases, since one of the fluorophoresattached to either the first or the second compound comes off and washesaway. An example of this type of reaction may include the first compoundbeing a protein, comprising fluorescent substituents attached thereto,and the second compound being a protease, which cleaves the protein intosmaller pieces.

The change in the fluorescence of the sample causes a change in the SPEFmeasurement. The rate of change of the fluorescence in the sample canthen be measured, which would be proportional to the rate of thereaction proceeding.

By comparing the measurements obtained from SPR and SPEF, one candetermine how fast the two compounds bind to each other and how fast thereaction between them proceeds.

Certain systems and methods of the present invention are alsoparticularly suited for measuring diffusion and reactivity of molecularreactions. Diffusion and reactivity can be measured by flowing at leasttwo interfacing fluid streams on a surface creating and relaxing surfacegradients as a result, at least one of the streams containingmacromolecules, the macromolecules interacting with the surface, whereinthe flow has a low Reynolds number so that the at least two streams donot mix and detecting the interactions. The system and method employ anassay that measures the surface diffusion of a macromolecule as itreacts with the substrate surface without altering the enzyme bylabeling. Specifically, gradient diffusion is measured, which contraststo self diffusion. Gradient (or mutual) and self diffusion are quitedifferent from one another, as self diffusion is strictly due to thermalenergy and gradient diffusion involves the relaxation of concentrationfluctuations (or gradients); both can be limited by strongerinteractions with the surface. This can specifically be done withmicrofluidic chips with three or five inlets flowing into a main channelin which the surface is covered by the protein substrate. More inletsare contemplated. Buffers are flowed down the outside lanes and enzymesthrough the middle, forming a stripe of enzyme down the middle of thesubstrate surface. The boundary between the enzyme lane and the buffersis kept relatively sharp by flowing at a high linear velocity (10-20cm/s). Initially the thickness profile of the surface will be flat, butonce enzyme flow begins, a trench will form, as the enzyme will cleavethe substrate adjacent to the middle lane. Since the enzyme solution inthe bulk is localized to the middle lane, any widening of this trenchmust be due to surface diffusion of the enzyme (i.e. relaxation of theenzyme concentration gradient) as it reacts away the surface boundprotein.

The system and methods may be either a “push” or “pull” mechanismwherein the push mechanism makes use of an acrylic flow cell. The flowcell serves as the housing for a chip, allowing an introduction offluids into the chip. The embodiment also contemplates a means to detectthe interactions, as provided above. An optical system to image theinteractions described may also be used. Possible methods for detectioninclude plasmon, fluorescence, Brewster angle or ellipsometric imaging.

The examples below provide more specific situations where suchmeasurements have taken place.

III. System

Disclosed is a system for quantifying the properties of molecules.Specifically, a system can be set up to perform and capture data fromSPR and SPEF simultaneously. FIG. 1 illustrates one embodiment of suchan apparatus. FIG. 1 illustrates a testing system 100 having a testingapparatus 200 and a computer 400. The testing apparatus 200 is incommunication with the computer 400 through a data link 202 to operateand report on the tests being run. The computer 400 sends commands tothe test apparatus 200 to control the test sequence and monitorparameters of the test. The test apparatus 200 sends data from the SPRand SPEF measurements to the computer 400 for storage and analysis.

FIG. 2 illustrates an embodiment of the testing apparatus 200 that issuitable for use in performing the simultaneous testing for SPR andSPEF. A laser 205 provides a source of light to operate the test. Thelaser can be monochromatic or it may generate various wavelengths oflight. Preferably, the laser is a 35 mW He—Ne laser. However, any lightsource can be used, such as a tunable light source or an omni-chromaticlight source, i.e., a source capable of emitting all wavelengths oflight simultaneously. The light is directed via mirrors 208, 212 througha neutral density filter 210 that controls the incident light intensityinto the rest of the components.

The beam of light passes next through a plane polarizer 215, such as aGlan-Thomson polarizer, and then through a spatial filter 220. Thespatial filter 220 gives the light a more homogeneous profile. The beamis then focused by a lens 225 into an expander/collimator 230 where thelight is expanded to the proper beam width and the light rays are madeparallel to one another.

The light is then directed into a test area 232 by a planar cylindricallens 235 at an angle below the critical angle. The critical angle is anangle below which all of the incident light 238 is reflected out asreflected light 242. The incident light is directed to a sample cell 300(shown in FIG. 3) by a hemi-cylindrical prism 240.

The incident light 238 passing through the prism 240 reflects off asurface 310 of the sample cell 300 and out of the prism 240. Thereflected light 242 then passes through a second planar cylindricalfocusing lens 250, which focuses the reflected light 242 onto a linearCCD array 260 which measures the intensity of the reflected light 242. Asecond neutral density filter 245 can also be used to attenuate thereflected light 242 to a level for which the photo detector is mostlinear or accurate. In the example illustrated in FIG. 2, a secondneutral density filter 245 is used with a CCD array 260, although anyphoto detection device can be used. Furthermore, the focusing planarlens 250 is used to focus light reflecting at a multitude of angles ontothe linear CCD array 260 in the embodiment illustrated in FIG. 2.However, it should be realized that a planar CCD array could be used aswell to aggregate the light reflected at various angles. The two methodsof vertically averaging, or aggregating the light reflected at thevarious angles, can be used but any other method of measuring theintensity of the total aggregated reflected light can be used. The lightmeasured by the CCD array 260 corresponds directly to any resonance thatoccurs with the surface plasmons and, therefore, is used to calculatethe SPR.

The SPEF is measured simultaneously with the SPR by a photo detector 280capable of detecting fluorescent emissions from fluorescent samples inthe sample cell 300. The illustrated embodiment uses a microscopeobjective lens 270 and a photomultiplier tube 280, but any other meansof measuring fluorescent emissions can be used without departing fromthe spirit of the invention.

Referring to FIG. 3, one embodiment of the sample cell 300 is describedfor performing the SPR and SPEF tests. Again, although one embodiment ofthe sample cell 300 is illustrated in FIG. 3, any number of variationscan be used. The illustrated embodiment includes a thin gold film 305attached to the glass slide 310. In some embodiments the glass slide 310is polished SF10 glass, but any glass can be used.

Some embodiments may also include another layer of a different metalbelow the thin gold film. The purpose of this second layer of metal isto provide better adhesion of the gold layer to the glass. A number ofdifferent metals can be used for this purpose, including, but notlimited to, chromium, molybdenum, vanadium, niobium, tantalum andmanganese. Preferably, the undercoat on the gold film includes chromium.

A functionalized anchor 320 is affixed to the thin film 305 and isdesigned to chemically react with other molecules that are to beattached to the thin gold film 305. The anchor may be in the form ofself-assembled monolayers. The anchor may be affixed to the thin film305 via a variety of different functional groups, such as, but notlimited to, thiols, sulfoxides, sulfones, thiosulfates, disulfides,polydisulfides, alkylsulfides and the like. The anchor then presents afunctional group, to which the first compound can be attached. Virtuallyany functionality can be presented by the anchor 320. In some cases, thefunctional group may be a reactive group such as an amine, carboxylate,sulfide or ester. In other instances, biological functionalities such aslipids, biotins, antibodies or ligand/receptor molecules may bepresented at the surface. Generally, this specific functionality willdetermine the type of static or dynamic measurements to be performed.

After a test compound is attached to the functional group of the anchor320, a solution comprising the sample compound is then contacted withthe combination of glass 310, thin metal layer 305, anchor 320, andlinked test compound. In some embodiments, the solution flows over theglass slide 310. In other embodiments, a lower glass slide 330 forms aflow area 340 between itself and the anchor 320 through which testsolution can flow into and out of the test cell 300.

The gap between the lower glass slide 330 and the upper glass slide 310,or the height of the flow area 340, will depend on the test performed.The larger the gap, the more solution can pass through. However theratio of the volume of the solution passing through the cell to thecontact area with the test compound decreases as the gap becomes larger.Those of skill in the art can determine how large a gap is suitable forthe type of test is being conducted. In certain embodiments, the gap isnot larger than 1 cm. In other embodiments, the gap is less than 5 mm,less than 1 mm, or less than 0.5 mm.

In certain embodiments a gasket of a certain thickness is used betweenthe glass slide 310 and lower glass slide 330 to create and hold thegap. The thickness of the gasket, then, determines the thickness of thegap. The gasket may be made of any number of materials, but preferablyit is made of such material that are inert towards the solutions and thecompounds used in the test. Examples include, but are not limited to,rubber, ceramic, silicone, TYGON, TEFLON and the like. Some embodimentspreferably use a 0.5 mm silicone gasket to determine the gap.

The entire assembly 342 is then attached to the hemi-cylindrical prism240 to generate the sample cell 300. In some embodiments the prism 240will be made of the same glass type as the glass slide 310. In oneembodiment, the glass is SF10 glass. Additionally, an index matchingfluid is placed between the prism 240 and the glass slide 310 to preventany refraction of laser light as it passes from the prism 240 to theglass slide 310. If an SF10 glass is used that has a refractive index ofabout 1.723, then one example of a liquid that can be used is a Series Mliquid having a refractive index of about 1.730 from CargilleLaboratories, Inc., 55 Commerce Road Cedar Grove, N.J. 07009.

A solution is then passed through the flow area 340 using any flowgenerating mechanism capable of providing flow, such as a peristalticpump or a gravity drain system, for example. Certain embodiments utilizea flow generating mechanism or means that provides steady and repeatableflow characteristics. An SPR of the sample cell 300 is established byactivating the laser 205 and directing the incident light at theappropriate angle such that SPR commences, and then the compounds (notshown) the user desires to have react with the anchor 320 are added tothe fluid. As mentioned above, as the compounds react with the anchor320, the refractive index of the test surface 320, 305, 310 changes,thereby changing the SPR reflection angle. In some embodiments, theangle of incidence is changed to determine which angle the resultantintensity of the reflected light 242 is weakest thereby indicating theSPR angle at any time, commonly referred to as scanning. This SPR angleis tracked by repeatedly scanning as compounds react with the test cellanchor 320 and because the SPR angle corresponds to the refractive indexof the sample cell test surface 320, 305, 310 at any time users candetermine what reactions have occurred by the changes of the SPR angle.

In the embodiment illustrated in FIG. 2, changing the angle ofincidence, or scanning, is not required as the planar focusing lenses235, 250 focus the light at various angles onto the test surface 320,305, 310 and then to the CCD array 260. Moreover, altering the angle ofreflected light 242 is not required in embodiments utilizing a planarCCD array that can aggregate the intensity of the reflected light 242over a range of angles. In one embodiment the range of angular widththat can be captured by the second planar focusing lens 250 isapproximately eight degrees. However, this range can be more or lesswithout departing from the invention. Typical SPR scanning techniquesshow a dip in the intensity of the reflected light at the angle whereresonance occurs. This angle changes as the refractive index of theanchor 320 surface changes from reactions with compounds in the fluidflowing through the test cell 300. To maintain the linearity of the SPRand SPEF readings, in embodiments not aggregating a range of angles ofthe reflected light 242, this angle is preferably tracked and accountedfor as the test continues. However, in embodiments, aggregatingreflected light 242 from a range of angles, the linearity between thetests is maintained as the total intensity of all the reflected light inthe range of concern is measured. This light energy varies as photonenergy is transferred to the surface plasmons and also to thefluorescent light emitting materials during SPEF.

As mentioned above, a computer 400 is used to control and monitor thetesting apparatus 200 for the SPR and SPEF procedures, as illustrated inFIGS. 1 and 4. The computer 400 fulfills several functions such ascollecting and processing data, data analysis, storage, and varioustypes of output. The computer 400 has several modules that fulfill thesefunctions. However, it should be noted that the computer 400 can havemore or less modules than are illustrated in FIG. 4. As can beappreciated by one of ordinary skill in the art, each of the modules caninclude various sub-routines, procedures, definitional statements andmacros. Each of the modules can be separately compiled and linked into asingle executable program. Therefore, the following description of eachof the modules is used for convenience to describe the functionality ofthe computer 400. Thus, the processes that are undergone by each of themodules may be arbitrarily redistributed to one of the other modules,combined together in a single module, or made available in a shareabledynamic link library.

The computer 400 has an input device 402 that allows the operator toenter commands the computer 400 will execute. For example, the inputdevice 402 may be a keyboard, rollerball, pen and stylus, mouse, orvoice recognition system. The input device 402 may also be a touchscreen associated with feedback provided by the computer 400. The usermay respond to prompts on the display by touching the screen. The usermay enter textual or graphic information through the input device.

The computer 400 also has an input/output module 405 for communicationwith the test apparatus 200 (FIGS. 2 and 4). The I/O module 405 providesfor one-way or two-way communication and may provide simultaneous orordered communication with the laser 205, the CCD array 260, thephotomultiplier tube 280, or any other components of the test apparatus200. The computer 400 may be in communication with the test apparatus200 via a direct connection or via a network connection. The networkconnection may be a local area network, a wide area network or any othertype of private or public network, such as the Internet. Alternatively,the computer 400 may be part of, or integral with, the test apparatus200.

Still referring to FIG. 4, the computer has a processor 410 forexecuting many or all of the functions of the computer 400. Theprocessor 410 may be any conventional general purpose single- ormulti-chip microprocessor such as a Pentium® processor, a Pentium®) Proprocessor, an 8051 processor, an MPS® processor, a Power PC® processoror an ALPHAS processor. In addition, the processor 410 may be anyconventional special purpose microprocessor such as a digital signalprocessor. The computer 400 also includes a storage medium 420 forstoring data or other information. The storage medium 420 can be anytype of computer data storage medium including, but not limited to, RAMmemory, DRAM memory, SDRAM memory, flash memory, ROM memory, EPROMmemory, EEPROM memory, registers, hard disk, a removable disk or CD-ROM.The storage medium 420 is coupled to the processor 410 such that theprocessor 410 can read information from, and write information to, thestorage medium 420. In the alternative, the storage medium 420 may beintegral with the processor 410. The processor 410 and the storagemedium 420 may reside in an Application Specific Integrated Circuit(ASIC).

As illustrated in FIG. 4, in addition to the modules described above,the computer 400 has special purpose modules that are directed to thetesting of SPR and SPEF. These include a reference module 430, ananalysis module 440 and an output module 450. In one embodiment thereference module 430 provides instructions for determining normalizedreadings of SPR and SPEF values rather than absolute values. Thus, thereference module 430 is configured to perform normalizing tasks, such asincorporating reference values into the data gathered from the SPR andSPEF procedures. The reference module 430 serves many data normalizingfunctions such as data normalization, test reference level determinationand reflectance normalization. As described below, some embodimentsutilize a normalized reflectance or an effective reflectance valuerather than the actual or absolute reflectance taken during SPR or SPEF.The information required to perform such normalization processes isstored in, or performed by, the reference module 430.

Another module illustrated in FIG. 4 is the analysis module 440. Theanalysis module 440 provides the routines or subroutines necessary toprocess the data collected from a SPR or SPEF test to determine theresults of the test in the format desired by the user. The analysismodule 440 may be a slave module or include a coprocessor operated bythe processor 410. Alternatively, it may be an independent moduleoperating separately from the processor 410. Moreover, the analysismodule 440 may be separate from the computer 400, such as in a separateprocessing device or in the testing apparatus 200.

The computer can also utilize the output module 450 to present feedbackto the operator. The output module 450 may be part of the input module402, such as in a touch screen to provide operational feedback as theoperator is operating the computer 400. The method of output depends onthe application and the desires of the operator and can be, for example,photo or electronic signals to be used by other devices, a plotter, avideo output, a printer, a graphing device, a speaker or any otheroutput device.

The various illustrative logical blocks, modules, and circuits describedin connection with the embodiments illustrated by FIG. 4 and describedherein may be implemented or performed with a general purpose processor,a digital signal processor (DSP), an application specific integratedcircuit (ASIC), a field programmable gate array (FPGA) or otherprogrammable logic device, discrete gate or transistor logic, discretehardware components or any combination thereof designed to perform thefunctions described herein. A general purpose processor may be amicroprocessor, but in the alternative, it may be any processor,controller, microcontroller or state machine. A processor may also beimplemented as a combination of computing devices, e.g., a combinationof a DSP and a microprocessor, a plurality of microprocessors, one ormore microprocessors in conjunction with a DSP core or any other suchconfiguration.

Reference values used in the testing process, and by reference module430, can be obtained at different stages. However, some embodimentsmeasure reference values of SPR and SPEF readings of the test samplecell 300 (FIG. 3) prior to passing any fluid across the test surface.FIG. 5 illustrates an exemplary normalization process 500 that can bepart of the reference module 430 and can be used to determine aneffective reflectance reading for the system. The process 500 begins ata start state 510 and moves to a first measuring state 520. At the firstmeasuring state 520, the surface reflectivity of the sample cell ismeasured over the range of TIR angles. Values of reflectance are storedto the storage 420 when no liquid is flowing through the sample cell toprovide a surface to air reading. This could be performed with a testsurface to air measurement as described. However, a reference fluid,such as water, a buffer or a standard solution, may be used as well.

After the reference values are measured and stored to the storage 420,the process 500 moves to a second measuring state 530 wherein the SPRtests are performed again using the same fluid that that will beeventually used during the SPR and SPEF test, such as a buffer, withoutthe compound to be tested. These values are again stored to be used in adetermining state 540. In the determining state 540, the individualvalues of the data points from the second measuring state 530 aredivided by their corresponding data points from the first measuringstate 520, and the results are recorded as the normalized reflectance oreffective reflectance signal. By dividing the fluid to surfacemeasurements with the air to surface measurements, an effectivereflectance rather than an actual reflectance is determined resulting ina relatively smooth set of results. By normalizing the data in this way,it is not necessary to measure the incident light to determine ameasured reflectance, which may be defined as the reflected lightintensity divided by the incident light intensity, thereby eliminatingthe need for additional components. FIG. 6 a illustrates a set ofsurface to buffer interface SPR data taken over a range of reflectionangles that has not been normalized and that is superimposed over a plotof reference data of surface to air readings over the same range ofangles. The surface to buffer data points drop dramatically between 56degrees and 58 degrees as resonance occurs while the reference datapoints remain relatively constant through this range. However, both setsof data are relatively erratic over the range of data and normalizationcan be utilized to counteract this effect. As illustrated in FIG. 6 b,the normalization of the surface to buffer data can result in a smootherset of data points over the measured range. In FIG. 6 b, the set ofreference data points appears as a straight line at an effectivereflectance value of 1, where the buffer to surface data appears as asmooth curve over the range measured.

However, the incident light may be measured and actual reflectance canbe measured if desired, or, in the alternative a reference reflectivitycan be measured by subtracting the reference data of the first measuringstate 520 from the reflectance data of the second measuring state 530.The process 500 then ends at a terminating state 550. By the process 500illustrated in FIG. 5, irregularities in the beam of light being usedcan be accounted for and the SPR results can be substantially repeatedregularly for consistent and reliable results.

In another embodiment of the invention, a system for quantifying theproperties of lateral diffusion and reactivity of adsorbedmacromolecules measured by microfluidic patterning of substrate surfacesis disclosed. Specifically, a system is disclosed to perform and capturedata from flowing, creating and relaxing surface gradients in at leasttwo interfacing fluid streams, at least one of the streams containingmacromolecules, the macromolecules interacting with the surface, whereinthe flow has a low Reynolds number so that the at least two fluidstreams do not mix. FIG. 13 illustrates one embodiment of such anapparatus. FIG. 13 illustrates a testing system having a testingapparatus similar to that provided above 1300 and can have a computer,as provided above. The testing apparatus 200 is in communication withthe computer 400 through a data link 202 to operate and report on thetests being run. The computer 400 sends commands to the test apparatus200 to control the test sequence and monitor parameters of the test. Thetest apparatus 200 sends data measurements to the computer 400 forstorage and analysis. Observation of the phenomenon can also be madevisually.

According to this embodiment of the invention, channel geometry can beestablished, as shown in FIG. 13, where two fluids are flowed inalternating lanes and gradients are created and relaxed. As can be seen,the fluid in the middle lane 1301 will be directed down the center ofthe channel with limited mixing with fluid in the outer lanes 1302.Because of the lack of inertial forces and turbulence in the system, theonly mechanism for mixing in the microchannel is the relatively slowprocess of diffusion. Also, because all lanes are flowing in a devicesuch as described herein, molecules that have diffused across laneboundaries are promptly swept downstream by the flow.

Buffers are flowed down the outside lanes 1302 and enzymes through themiddle 1301, forming a stripe of enzyme down the middle of the substratesurface. The boundary between the enzyme lane and the buffers is keptrelatively sharp by flowing at a high linear velocity (10-20 cm/s).Referring to FIG. 15, initially the thickness profile of the surfacewill be flat as no cleavage has yet occurred. Once the enzyme flowbegins, a trench will form, as the enzyme will cleave the substrateadjacent to the middle lane. Since the enzyme solution in the bulk islocalized to the middle lane, any widening of this trench will be due tosurface diffusion of the enzyme as it reacts away the surface boundprotein. Measurement of surface diffusion of the enzyme is indirect asthe presence of diffusing unlabeled enzyme is shown by loss of labeledsubstrate. This is an important aspect of the technique because theactual observable is not the enzyme itself and thus although the trenchformation can be imaged by methods that are completely label free, suchas ellipsometric, brewster angle or surface plasmon imaging. Theindirect nature of the technique also permits use of a label (i.e. alabeled substrate). As a result, widening of the region of reducedintensity indicates surface diffusion.

The embodiment measures gradient diffusion, in contrast to selfdiffusion coefficients measured by earlier TIR-FRAPP experiments.Although the two phenomena of gradient (or mutual) and self diffusionare quite different from one another, as self diffusion is strictly dueto thermal energy and gradient diffusion involves the relaxation ofconcentration fluctuations (or gradients), both can be limited bystronger interactions with the surface. Thus, the effect of an enzymethat is less active due to limited mobility should be seen regardless ofwhether we measure gradient diffusion or self diffusion.

FIG. 17 shows a device of the invention, a microfluidic chip. The chipcomprises a PDMS piece 1701 with an embedded channel geometry created bysoft lithography techniques; a channel geometry with multiple inputchannels 1702 combined into a single straight channel; 1 mm diametercircular holes in the PDMS piece at each inlet and outlet 1705 to allowfor the delivery of fluids to and from the chip; a glass slide 1703sealed against the PDMS piece; a flow cell (not shown) composed ofmultiple inputs and a single output matching those of the PDMS piece; arectangular cutout (not shown) for placement of the microfluidic chip;an aluminum clamp (not shown) which may fit either over a prism adjacentto the glass slide 1704 or directly onto the glass slide and serves toapply pressure to the chip ensuring a tight seal; a rectangular acryliccover plate 1707 with holes drilled through the plate that are matchedup with the inlets on the chip and serve as fluid reservoirs 1708 foreach of the inlets; a polished smooth bottom side of the plate thatseals tightly against the PDMS portion of the chip; a siphon system 1709to keep the reservoirs filled and an acrylic base plate 1706 withcomparable dimensions to the cover plate.

The embodiment is of a “pull” configuration, which directly deliversfluids to the chip without any external connectors. The system is notclosed, but a closed system of the invention may also be envisioned. Aclosed system may be more adaptable to imaging systems, as providedabove, which require a prism to be coupled to the chip. The siphon onlyfills the reservoirs containing buffers. The reservoirs containingenzyme solutions are filled by individual syringes attached to a singlesyringe pump. A glass microscope slide presents the appropriatesubstrate surface, such as a covalently bound protein layer.

The base plate serves the same purpose as the clamp in a push setup asit tightens down to seal the chip as well as providing a window forobservation through a microscope. Solutions comprising enzyme moleculesand pure buffer flowed in alternate inlets of the flow cell at lowReynolds number allow for creation of multiple lanes of fluids notinteracting with each other but without any physical barrier. Detectionis with fluorescence microscopy on an inverted microscope equipped witha Hg light source, red light filter and a CCD camera. The systemrequires a non-fluorescent protease flowing over a fluorescently labeledsubstrate surface. Loss of intensity indicates cleavage.

EXAMPLES

The following examples are used to illustrate certain embodiments of thepresent invention and are not meant to be limiting.

Example 1 Biotin/Avidin/Sandwich Experiment

A schematic of an experimental setup is shown in FIG. 3. In thisexample, a 35 mW He—Ne laser was directed by a series of mirrors througha neutral density filter (used to control incident light intensity) andinto the optical train. The beam passed through a Glan-Thompsonpolarizer to ensure p-polarized light for the SPR experiment. A spatialfilter removed stray light from the beam, yielding a more homogeneousprofile, followed by a beam collimator/expander which expanded it to theappropriate diameter (˜2.5 cm.).

The widened beam was focused onto the sample cell by a verticallymounted planar cylindrical lens, as in the scanning angle reflectometryapparatus employed by Leermakers et al. Leermakers, F. A. M.; Gast, A.P., Macromolecules, 1990, 24(3), 718-730. An entire SPR spectrum canthus be captured at once on the CCD. A horizontal spectrum over anangular width of ˜8° was captured. This differs from the approach usedby Lieberman et al. (Liebermann, T.; Knoll, W., Colloids Surf A,Physicochem. Eng. Aspects 2000, 171(1-3), 115-130).

The reflected beam was “vertically averaged” by a horizontally mountedcylindrical lens and directed onto the CCD element of a 1D CCD, whichrecorded the reflectivity data. Fluorescence was measured from behindthe sample cell. A 5× microscope objective lens collected light emittedfrom the center of the sample cell and a PMT recorded a photon count.Both the PMT and camera were connected directly to a PC computer anddata was recorded using Labview software.

A diagram of the sample cell 300 is also shown in FIG. 3. In thisexample, a thin is 50 nm gold film with a 2 nm chromium undercoat wasevaporated onto a polished SF10 glass (n=1.723) slide (Schott GlassTechnologies). The surface was functionalized by means of a thiol anchorcarrying the selected reactive group. Another glass slide was sandwichedtogether with the first, with the gap between them defined by a siliconegasket (thickness 0.5 mm). A solution containing the analyte was thenflowed through the cell by means of a peristaltic pump.

The entire assembly was mounted onto an SF10 glass (n=1.723)hemi-cylindrical prism. Index matching is accomplished via a Series Mindex matching liquid (n=1.730) from Cargille Laboratories. Thisprovided a close but not perfect match to the SF10 glass and as aresult, an interference pattern was observed on the CCD image. Theeffects of this pattern were eliminated by vertically averaging theimage using a second focusing lens 250 (FIG. 2) just in front of thecamera.

Self assembled monolayers of propionic acid were prepared by dipping thegold-coated slides into a 3-mercaptopropionic acid solution (200 μL/100mL) for 30 min. The carboxyl end groups of the immobilized hydrocarbonchains were activated for peptide bond formation using EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride)/NHS(N-hydroxysuccinimide) chemistry. Slides were immersedin a solution of 40 mg/100 mL EDC and 60 mg/100 mL NHS in reactionbuffer (2-(N-morpholino)ethanesulfonic acid 20.62 g/L, NaCl 29.2 g/L)for 1 hr. The EDC/NHS step produced a water stable ester able to reactwith primary amines. Biotinylated surfaces were produced by reactionwith biocytin hydrazide (Molecular Probes B-1603, 10 mg/100 mL for 2hours) in reaction buffer. Monolayers of covalently bound fluorescentlytagged bovine serum albumin (BSA) were formed by reaction with TexasRed/BSA conjugates (Molecular Probes—A23017, 2.5 mg/mL for 2 hours) inreaction buffer. In either case, the slides remained overnight inprotein or biocytin hydrazide solution to ensure complete reaction.

Avidin-Texas Red conjugates (A-820) were purchased from MolecularProbes. Biotin in solution was conjugated to BSA in a manner identicalto that used to biotinylate the surface. Carboxy-side chains of BSA (10mg/100 mL) were activated by reaction with EDC (400 mg/100 mL) and NHS(600 mg/100 mL) for one hour and then covalently linked to the aminegroup in biocytin hydrazide (5 mg/100 mL). Conjugates were separatedfrom unreacted biotin using gel filtration chromatography. The proteinused was B. lentus subtilisin enzyme. All proteins were assumed to havea refractive index of n=1.57. Jung, L. S. et al. Langmuir 1998, 14 (19),5636-5648.

A typical “raw” reflectivity plot is shown in FIG. 7 a. The gold/bufferreflectivity curve was normalized on the gold/air signal at the sameangular range. This image processing step allowed the effects ofinhomogeneities in the beam profile caused either by the laser or thesurface to be removed. Normalized curves are shown in FIG. 7 b.

As illustrated in FIG. 8, this experiment included binding a monolayerof biotin to a functionalized layer of hydrocarbons. The monolayer offluorescently labeled avidin was allowed to bind the biotin (FIG. 8 a).Based on the Liebermann et al. (Colloids Surf A, Physicochem. Eng.Aspects 2000, 171(1-3), 115-130) results, the SPR and SPEF signals wereproportional to one another. The use of a planar cylindrical lens tofocus a fan of laser light onto the surface further simplified thisanalysis. In a conventional single angle kinetics experiment, the amountof energy transfer to the surface plasmon wave changed through thecourse of the experiment. This detracted from the linearity of thecorrelation between the SPR and SPEF signals. Liebermann et al., supra,discussed this effect and stated that it must be accounted for duringthe analysis. However, in the present experiments, light was impingingon the surface at a range of angles and thus the intensity transfers tothe surface plasmon wave remained constant throughout the experiment.Thus, no further signal processing was necessary to linearly correlatethe SPR and SPEF measurements.

In the next step, labeled and unlabeled avidin were successively addedto the monolayer of biotin (FIG. 8 b). The SPR signal rose throughoutboth additions due to a continued increase in protein layer thickness;however the SPEF signal only rose following addition of the labeledprotein. Thus, the two signals were used to simultaneously measurebinding of the labeled and unlabeled species.

The same idea was used to distinguish the presence of two separateproteins on the surface in the second part of a “sandwich” experiment(FIG. 8 c). The first layer consisted of unlabeled avidin and thus norise in SPEF signal was observed. A fluorescently labeled biotin-BSAconjugate was then added to this layer. The rise in SPEF signalindicated the formation of the second layer. Once again, the amounts ofthe two proteins on the surface were differentiated by use of the SPRand SPEF signals in tandem.

The results from these three experiments are shown in FIGS. 9-11. InFIG. 9, fluorescently labeled avidin was flowed over a biotin monolayerand bound forming a 31±3 Å thick layer. SPR and SPEF signals wereessentially identical. The inset clearly shows the linearity of thesignals. Another advantage of the tandem experiment is that it discountsthe need for an independent calibration of the fluorescence signal. Thetwo signals can be calibrated with one another and an average layerthickness determined.

An example of an incomplete monolayer is shown in FIG. 10. Labeledavidin was allowed to bind to a thickness of ˜18 Å. Unlabeled avidin wasthen added and allowed to complete the monolayer (FIG. 10 a). Thissecond step illustrates the separation of the two components, as the SPRsignal rose but the SPEF signal remained unchanged. The correspondingcontrol experiment is shown in FIG. 10 b. In this case, labeled avidinwas added in both steps demonstrating no distinction between SPR andSPEF signals.

Finally in FIG. 11, the same idea was applied to the distinction of twounrelated proteins. Fluorescently labeled BSA was passed over anunlabeled avidin layer and bound to a thickness of ˜10 Å (FIG. 11 a). Afluorescence signal was only detected in the BSA step and thus theamounts of the two proteins on the surface at any given time weredetermined as shown in the inset. In the corresponding control (FIG. 11b), unlabeled BSA was added and no fluorescence signal was detected ineither step.

Example 2 Measurement of Enzyme Kinetics

Enzymes

A goal of the present experiment in developing this technique was tosimultaneously measure the adsorption and reaction kinetics of an enzymeinteracting with a substrate surface. The model substrate for thisexperiment was fluorescently labeled BSA. The enzyme was the serineprotease subtilisin. Subtilisin adsorbs to and cleaves BSA from thesurface.

For this study, variants of Bacillus lentus subtilisin (BLS) were used.This enzyme differs from the commercially available Subtilisin BPN′(BPN) at 103 of 269 residues. Kuhn et al., Biochemistry 1998, 37,13446-13452. BLS (MW=27 kD) is a serine protease with the characteristicSer(221), His(64), Asp(32) catalytic triad in its active site. Thereference enzyme for the experiment, which was labeled BLSv1, containedthree additional mutations to the BLS structure: N76D (substituteasparagine 76 with aspartic acid), S103A (substitute serine 103 withalanine), and V1041 (substitute valine 104 with isoleucine). The effectsof surface charge variations were investigated by studying enzymes withadditional single charge mutations on the surface of BLSv1.

The five variants were thus labeled: BLSv1 (the reference enzyme),BLSv1-Q109R (substitute neutral glutamine 109 with positively chargedarginine), BLSv1-G159D (substitute neutral glycine 159 with negativelycharged aspartic acid), BLSv1-Q206R (substitute neutral glutamine 206with positively charged arginine) and BLSv1-Q206E (substitute neutralglutamine 206 with negatively charged glutamic acid). BLSv1-Q109R andBLSv1-Q206R are referred to as positive mutants, and BLSv1-G159D andBLSv1-Q206E as negative mutants. In each case, the mutation is far fromthe active site.

Coulombic and Poisson Boltzmann surface charge calculations show thatBLS is a polar molecule, in which the “front” surface (containing theactive site) is neutral to negatively charged and the “back” side has amore positive character. The four mutants of the reference enzyme are“front side” mutants. All enzymes were provided by GenencorInternational (Palo Alto, Calif.). The enzyme stock solutions were usedas received.

Buffer Solutions

High ionic strength buffer (2 mM sodium carbonate+˜15 mM sodium sulfate,conductivity=3.2 mS/cm) and low ionic strength buffer (2 mM sodiumcarbonate) were used for the enzyme reaction experiments. Experimentswere run at pH 10, the optimum pH for BLS activity.

Substrate Surfaces

Immobilized substrate surfaces were prepared on glass slides (SchottGlass Technologies; SFIO glass material; dimension 1 in.×1 in.×1 mm). Athin chromium (2 nm) undercoat followed by a 50 nm gold film weredeposited on the glass slides by thermal evaporation using an EdwardAuto 302 vacuum coater. The gold-coated slide glasses were dipped in3-mercaptopropionic acid solution (200 μL/100 mL) for 30 min. This thiolcoating produces a self assembled monolayer with acidic surface groups.The slides were then immersed in a solution composed of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (40 mg/100mL) and N-hydroxysuccinimide (60 mg/100 mL) in a reaction buffer(2-(N-morpholino)ethanesulfonic acid 20.62 g/L, NaCl 29.2 g/L) for 1 hrto activate the surface for attachment of the protein substrate. A 2.5mg/mL solution of Texas Red tagged bovine serum albumin (MolecularProbes) in the reaction buffer was added via a transfer pipet directlyonto the gold surface and the surface linking reaction allowed toproceed for two hours, creating a surface bound layer of BSA. Prior toeach experiment, the substrate surfaces were rinsed with Milli-Q waterand then dried by a gentle air stream. After the experiment, the goldfilms were removed by acid (70% hydrochloric acid+30% nitric acid), anda new gold layer was re-deposited for further experiments.

Enzyme Inhibition

Subtilisin is inhibited by addition of phenylmethylsulfonyl fluoride(PMSF). A 100 mM solution in ethanol was diluted to 2 mM in MES buffer,pH 5.5, and added to subtilisin stock solution (1 mg/mL) at 20 vol % andthen incubated at room temperature for 30 min.

SPR-SPEF Measurement

Details of the SPR/SPEF apparatus are described herein. A diagram of thesample cell is shown in FIG. 3. In this example, the flow channel wasmade of two glass slides with a silicone gasket insert (0.5 mmthickness). Buffer and enzyme solution were flowed at wall shear ratesof 400 s⁻¹. Bulk enzyme concentrations were 2 μg/mL. Angular SPRprofiles and fluorescence light intensities were measuredsimultaneously. The total protein (BSA+enzyme) layer thickness wasobtained from the angular SPR profile using a Fresnel calculation. Theamount of fluorescently labeled BSA was obtained from the fluorescencesignal intensity. The linearity of the two signals allows one todetermine the amount of adsorbed enzyme from their difference, asdescribed herein.

Strength of Association—PMSF Inhibited Enzyme Experiment

In the next experiment, the association between the enzyme and thesurface was examined further by comparing the adsorption properties ofthe five variants using a PMSF inhibited enzyme experiment. PMSF(phenylmethylsulfonyl fluoride) reacts with the serine in the catalytictriad and renders the enzyme 90% inactive. The nearly inactive enzymewas useful for studying adsorption onto an intact BSA monolayer, assubstrate hydrolysis was severely limited. Sample SPR results are shownin FIG. 12. Initially, buffer was passed over the surface to establish asurface plasmon. Following introduction of inhibited enzyme, the totalsurface layer thickened, as shown by the rise in SPR signal. Thedisplacement in the signal was indicative of the amount of adsorbedenzyme. Once a steady level of adsorption was reached, the enzymesolution was replaced by buffer. Active enzyme was subsequently passedover the surface to verify the presence of BSA through its loss tohydrolysis.

Reactivity

The reactivity was analyzed using time-course SPEF data. Thefluorescence signal was calibrated using the initial high fluorescenceas a reference point with 100% substrate. It was also noted thatfollowing completion of the reaction, the surface was free of substrate(this was confirmed by examining the location of the minimum of the SPRspectrum). The endpoint, therefore, corresponded to 0% substrate. Thistime-course data was converted to a more intuitive velocity vs.substrate concentration curve. The instantaneous rates of change wereobtained by taking slopes along the curve, with each point correspondingto a reaction velocity. A sample reaction velocity vs. substrate curvewas generated in this way. The early time (high substrate concentration)data was removed from the curve so as not to include artifacts resultingfrom the ten second fill time of our sample cell in the analysis.

In Situ Adsorption

The SPEF data alone provided information on reactivity, as shown in theprevious section. It was combined with SPR data to track enzymeadsorption as it hydrolyzed the BSA layer. A plot of enzyme adsorptionas a function of substrate concentration is obtained by simply matchingthe beginning and end SPEF and SPR signals and then taking a differencesignal. Since SPR measured total protein (enzyme+BSA) layer thicknessand the SPEF isolated BSA, their difference was the amount of enzymeadsorbed on the surface. The curve is divided into high and lowsubstrate concentration regions. The beginning of the low concentrationregime was delineated as the point where enzyme adsorption has reached aplateau and adsorption kinetics were no longer limiting. In this study,attention was focused on the low concentration (0-25% substrate) region,the same conditions that were studied in the solution assays.

Example 3 A Microfluidic Chip for Measuring Diffusion and Reactivity

A microfluidic chip was created for quantifying the properties oflateral diffusion and reactivity of adsorbed macromolecules measured bymicrofludic patterning of substrate surfaces, as shown in FIG. 17. Thechip comprises a PDMS define piece (25 mm×75 mm×˜3 mm thickness) with anembedded channel geometry created by soft lithography techniques; achannel geometry with multiple input channels combined into a singlestraight channel; a channel geometry with 100-200 μm channel widths and50 μm channel thicknesses; 1 mm diameter circular holes in the PDMSpiece at each inlet and outlet to allow for the delivery of fluids toand from the chip; a glass slide of dimensions 25 mm×75 mm×1 mm sealedagainst the PDMS piece; a flow cell composed of multiple inputs and asingle output matching those of the PDMS piece; a rectangular cutout ofdimensions (25 mm×75 mm×4 mm) for placement of the microfluidic chip; analuminum clamp which may fit either over a prism adjacent to the glassslide or directly onto the glass slide and serves to apply pressure tothe chip, ensuring a tight seal; a rectangular acrylic cover plate (witha base of dimension 2.75″×1.25″ and a thickness of 1.25″) with holesdrilled through the plate that are matched up with the inlets on thechip and serve as fluid reservoirs for each of the inlets; a polishedsmooth bottom side of the plate that seals tightly against the PDMSportion of the chip; a siphon system to keep the reservoirs filled; anacrylic base plate with comparable dimensions to the cover plate and athickness of no more than 0.2″.

Example 4 Creation of an Assay to Measure Surface Diffusion-Three andFive Lane Substrate Surfaces

Surfaces are prepared following the methods described by Gaspers et al.(16). Cleaned glass microscope slides (25 mm×37.5 nm×1 mm) are soaked inacetone for 10 minutes and then placed in a 0.1% v/v solution of3-amino-propyltriethoxysilane (ATES) in acetone, and incubated at 37° C.for 30 minutes. Carboxy-side chains of Texas Red/BSA conjugates(Molecular Probes—A23017, 0.5 mg/mL) were activated by reaction with EDC(20 mg/5 ml) and NHS (30 mg/5 ml) in cross linking buffer for one hour.The slides were then brought into contact with 1 mL of the activatedTexas Red/BSA solution for 48 hours at room temperature. The wettingproperties of the amine-terminated surface ensured complete coverage ofthe slide by the protein solution. BSA coated slides were stored incarbonate buffer at 4° C.

Creation of Buffers

Carbonate buffers are as described previously. PBS buffer was preparedat pH 7.4 ([phosphate]=10 mM; [NaCl]=150 mM). Enzymes described aswildtype (WT) are BLSv1 (as stated previously). The GG36 enzyme isSubtilisin BLS (34). All other enzymes are single amino acidsubstitutions of BLS.

Flow Schemes

Three and five inlets were used in a microfluidic chip. In allexperiments, flow was pressure driven. Positive pressure to the systemto “push” the fluids through the chip was initially chosen. The flowscheme is shown in FIG. 16. The five lane experiments were all conductedusing negative pressure, or in the “pull” configuration, showndiagrammatically in FIG. 17. In this case, no connections are necessaryas the chip is filled directly from reservoirs and pulled from a singleoutlet. The buffer reservoirs were kept filled using a siphon and theenzyme reservoirs were pumped full from a syringe pump.

Three Lane Experiment

The results of a three lane experiment with enzyme flowed down themiddle lane are shown in FIG. 18. The beginnings of trench formation arefirst seen after about 2 minutes. As the intensity in the middle regiondrops at later time points, the trench deepens with the passage of time.Interestingly, the trench also widens. FIG. 18 b, shows the intensityprofiles across the channel. The shaded area indicates the originalenzyme lane width. It is clear that after almost two hours of enzymeflow, the trench has widened considerably beyond the original lane. Thisis a clear indication that the enzyme is diffusing along the surface asit cleaves the substrate.

Five Lane Experiment

Five lane experiments more readily show reaction and diffusioncharacteristics of two variants simultaneously. In the five laneexperiments, different enzyme variants were flowed in lanes 2 and 4. Incontrol experiments however, as shown in FIG. 19, because of sharp turnsat the inlets, secondary flows led to a small degree of mixing and whatappeared to be a “flaring out” of the lanes. In order to keep the lanessharp, the structure of the junction was altered as shown in FIG. 19 b.The inlet streams were now “guided” into the main channel by the moresmoothed curved lines. As seen in FIG. 19 c, a marked improvement wasachieved in the sharpness of the lanes and the “flaring” effect in thelanes was effectively eliminated.

Enzymes were then run through the lanes, as shown in FIG. 20. In thiscase, the enzymes used were GG36, the wildtype subtilsin for thisexperiment, and the G100R mutant. The experiment was run using low ionicstrength buffer and it is clear that the wildtype is more reactive andfaster diffusing than the G100R mutant. This corresponds well withprevious adsorption data, because in cases where the enzyme is verystrongly adsorbed to the surface, the reaction rate is reduced.

CONCLUSION

Thus, those of skill in the art will appreciate that new devices andmethods used for quantifying the properties of molecules, andspecifically, measuring the diffusion and rate of reactions, aredisclosed that employ the simultaneous use of SPR and SPEF andmicrofludics.

One skilled in the art will appreciate that these methods are and may beadapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The devices, methods, andprocedures described herein are presently representative of preferredembodiments and are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention and are defined by the scope of the claims.

It will be apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention.

Those skilled in the art recognize that the aspects and embodiments ofthe invention set forth herein may be practiced separate from each otheror in conjunction with each other. Therefore, combinations of separateembodiments are within the scope of the invention as claimed herein.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions indicates the exclusion ofequivalents of the features shown and described or portions thereof. Itis recognized that various modifications are possible within the scopeof the invention claimed. Thus, it should be understood that althoughthe present invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are within the following claims.

1. A device for quantifying the properties of molecules by measuringsurface plasmon resonance and fluorescence of a sample, comprising: alight source capable of directing a beam of light at a sample cell,wherein said sample cell comprises a first compound bound to a metallicsurface; a first detector for measuring the surface plasmon resonancefrom said sample cell; a second detector for measuring the fluorescenceintensity from said sample cell; and a module for calculating thecatalytic activity of said enzyme from said surface plasmon resonancemeasurement and said fluorescence intensity measurement.
 2. The deviceof claim 1, wherein said metallic surface comprises gold.
 3. The deviceof claim 1, wherein said first compound is bound to SAMS (self-assembledmonolayers) on said metallic surface.
 4. The device of claim 1, whereinsaid sample cell comprises a prism.
 5. A system for quantifying theproperties of molecules by determining the rate of catalytic activity ofan enzyme; comprising: a light source capable of directing a beam oflight at a sample cell, wherein said sample cell comprises a fluorescentcompound bound to a metallic surface; a first detector for taking asurface plasmon resonance measurement of said sample cell as an enzymeis contacted with said compound; a second detector for taking afluorescence intensity measurement of said compound as said enzyme iscontacted with said compound; and a module for calculating the catalyticactivity of said enzyme from said surface plasmon resonance measurementand said fluorescence intensity measurement.
 6. The system of claim 5,wherein said metallic surface comprises gold.
 7. The system of claim 5,wherein said first compound is bound to self-assembled monolayers onsaid metallic surface.
 8. The system of claim 5, wherein said firstdetector is a reflectance detector, which detects light reflected fromsaid sample cell.
 9. The system of claim 8, wherein said first detectoris a CCD detector.
 10. The system of claim 8, wherein the angle saidreflected light makes with the normal of said sample cell issubstantially equal to the angle said beam of light from said lightsource makes with the normal of said sample cell.
 11. The system ofclaim 10, wherein the sum of said angle said reflected light makes withthe normal of said sample cell and said angle said beam of light fromsaid light source makes with the normal of said sample cell is less than180°.
 12. The system of claim 10, wherein said first detector detectsany variations in said angle said reflected light makes with the normalof said sample cell.
 13. The system of claim 5, wherein said seconddetector is a fluorescence detector, which detects fluorescence fromsaid sample cell.
 14. The system of claim 5, wherein said seconddetector is located on the opposite side of said sample cell as saidlight source.
 15. The system of claim 5, wherein said compoundfluoresces after being illuminated by said light.
 16. The system ofclaim 5, wherein said enzyme fluoresces after being illuminated by saidlight.
 17. The system of claim 5, wherein said light is monochromatic.18. The system of claim 5, wherein said light source is a laser lightsource.
 19. The system of claim 5, wherein said module comprises amicroprocessor.
 20. The system of claim 5, wherein said module comprisesa memory.
 21. The system of claim 5, wherein said module comprises acomputer implemented instructions.
 22. A method for measuring diffusionand reactivity comprising a) flowing at least two interfacing fluidstreams, at least one stream containing macromolecules, themacromolecules interacting with the surface, wherein the flow has a lowReynolds number so that the at least two fluid streams do not mix; b)creating and relaxing surface gradients from step (a); and c) detectingdiffusion and reactivity.
 23. The method according to claim 22, whereinthe at least two fluid streams comprises three fluid streams.
 24. Themethod according to claim 22, wherein the at least two fluids streamscomprises five fluid streams.
 25. The method according to claim 22,wherein the detecting step includes at least one of fluorescencemicroscopy, plasmon imaging, ellipsometric imaging, brewster anglemicroscopy or total internal reflection microscopy.
 26. The methodaccording to claim 22, wherein the surface comprises plastics, polymers,SAMS, lipid bilayers, glass, transparent materials, reflectivematerials, gold, biomaterials or biodegradable materials or acombination thereof.